Despite the success of tyrosine kinase-based cancer therapeutics, for most solid tumors the tyrosine kinases that drive disease remain unknown, limiting our ability to identify drug targets and predict response. Here we present the first large-scale survey of tyrosine kinase activity in lung cancer. Using a phosphoproteomic approach, we characterize tyrosine kinase signaling across 41 non-small cell lung cancer (NSCLC) cell lines and over 150 NSCLC tumors. Profiles of phosphotyrosine signaling are generated and analyzed to identify known oncogenic kinases such as EGFR and c-Met as well as novel ALK and ROS fusion proteins. Other activated tyrosine kinases such as PDGFRalpha and DDR1 not previously implicated in the genesis of NSCLC are also identified. By focusing on activated cell circuitry, the approach outlined here provides insight into cancer biology not available at the chromosomal and transcriptional levels and can be applied broadly across all human cancers.

EGF receptor ͉ c-Met ͉ oncogene dependence ͉ proteomics ͉ stable isotope labeling with amino acids in cell culture

To ensure survival in the face of genomic insult, cells have evolved complex mechanisms to respond to DNA damage, termed the DNA damage checkpoint. The serine/threonine kinases ataxia telangiectasia-mutated (ATM) and ATM and Rad3-related (ATR) activate checkpoint signaling by phosphorylating substrate proteins at SQ/TQ motifs. Although some ATM/ATR substrates (Chk1, p53) have been identified, the lack of a more complete list of substrates limits current understanding of checkpoint pathways. Here, we use immunoaffinity phosphopeptide isolation coupled with mass spectrometry to identify 570 sites phosphorylated in UV-damaged cells, 498 of which are previously undescribed. Semiquantitative analysis yielded 24 known and 192 previously uncharacterized sites differentially phosphorylated upon UV damage, some of which were confirmed by SILAC, Western blotting, and immunoprecipitation/ Western blotting. ATR-specific phosphorylation was investigated by using a Seckel syndrome (ATR mutant) cell line. Together, these results provide a rich resource for further deciphering ATM/ATR signaling and the pathways mediating the DNA damage response.DNA damage ͉ mass spectrometry ͉ phosphorylation M aintaining the integrity of the genome is of utmost importance for cellular survival. For this reason, cells have evolved complex mechanisms to inhibit cell cycle progression in response to genomic insult, termed the DNA damage checkpoint (1). Activating checkpoint mechanisms gives cells time to repair or bypass the damage using specialized DNA polymerases or, in cases of high levels of damage, to activate apoptotic pathways (2). Elucidating pathways involved in checkpoint activation and maintenance continues to be an active area of research.A family of phosphoinositol-3-phosphate kinase-like kinases are critical to the proper function of the DNA damage checkpoint. The two central kinases involved are ataxia telangiectasiamutated (ATM) and ATM and Rad3-related (ATR). This kinase family also includes DNA-dependent protein kinase (DNA-PK) and a more recently discovered member of the family, SMG1 (3). These kinases are activated in response to DNA damage and subsequently phosphorylate targets responsible for such diverse activities as blocking cell cycle progression, coordinating DNA repair activities, and affecting transcription of DNA damage response genes. ATR is activated in response to a variety of damaging agents: UV light, alkylating agents such as methyl methanesulfonate (MMS), and chemical inhibitors of DNA replication such as aphidicolin and hydroxyurea (4, 5). ATM, however, is primarily involved in the response to double-strand breaks, such as those caused by gamma irradiation (IR) (6). Deficiency in ATM/R, as well as other components of the DNA damage checkpoint, has been found to cause debilitating diseases such as ataxia telangiectasia (ATM mutants), Fanconi's anemia, Seckel syndrome (ATR mutants), and the avoidance of checkpoint activation to allow cancer progression.In response to DNA damage, ATM/R phosphorylate checkpoint k...

Proteomic studies of post-translational modifications by metal affinity or antibody-based methods often employ data-dependent analysis, providing rich data sets that consist of randomly sampled identified peptides because of the dynamic response of the mass spectrometer. This can complicate the primary goal of programs for drug development, mutational analysis, and kinase profiling studies, which is to monitor how multiple nodes of known, critical

Protein phosphorylation plays a central role in creating a highly dynamic network of interacting proteins that reads and responds to signals from growth factors in the cellular microenvironment. Cells of the neural crest employ multiple signaling mechanisms to control migration and differentiation during development. It is known that defects in these mechanisms cause neuroblastoma, but how multiple signaling pathways interact to govern cell behavior is unknown. In a phosphoproteomic study of neuroblastoma cell lines and cell fractions, including endosomes and detergent-resistant membranes, 1622 phosphorylated proteins were detected, including more than half of the receptor tyrosine kinases in the human genome. Data were analyzed using a combination of graph theory and pattern recognition techniques that resolve data structure into networks that incorporate statistical relationships and protein-protein interaction data. Clusters of proteins in these networks are indicative of functional signaling pathways. The analysis indicates that receptor tyrosine kinases are functionally compartmentalized into distinct collaborative groups distinguished by activation and intracellular localization of SRC-family kinases, especially FYN and LYN. Changes in intracellular localization of activated FYN and LYN were observed in response to stimulation of the receptor tyrosine kinases, ALK and KIT. The results suggest a mechanism to distinguish signaling responses to activation of different receptors, or combinations of receptors, that govern the behavior of the neural crest, which gives rise to neuroblastoma.

Additional informationPeer review information Nature thanks the anonymous reviewers for their contribution to the peer review of this work.

Alkylating agents, such as methyl methanesulfonate (MMS), damage DNA and activate the DNA damage checkpoint. Although many of the checkpoint proteins that transduce damage signals have been identified and characterized, the mechanism that senses the damage and activates the checkpoint is not yet understood. To address this issue for alkylation damage, we have reconstituted the checkpoint response to MMS in Xenopus egg extracts. Using four different indicators for checkpoint activation (delay on entrance into mitosis, slowing of DNA replication, phosphorylation of the Chk1 protein, and physical association of the Rad17 checkpoint protein with damaged DNA), we report that MMS-induced checkpoint activation is dependent upon entrance into S phase. Additionally, we show that the replication of damaged double-stranded DNA, and not replication of damaged single-stranded DNA, is the molecular event that activates the checkpoint. Therefore, these data provide direct evidence that replication forks are an obligate intermediate in the activation of the DNA damage checkpoint.

Background: Identification of ubiquitin ligase substrates remains an unmet challenge. Results: Two proteomic strategies were used to identify novel substrates of the E3 ligase HRD1. Conclusion: These methods identified populations of substrates enriched for potential targets of endoplasmic reticulumassociated degradation. Significance: This approach should be broadly useful for E3 ligase substrate identification, and the identified substrates provide insight into the role of HRD1 in disease.